Parallel comparison of 5 different DC methods shows comparable average DCA expression rates and upregulation after conversion of MNC to DC in AML, MDS or

healthy samples

It has been shown that in 6%-60% of healthy, AML or MDS cases, less than 10% of DC could be generated. In order to find at least one DC-generating method for every individual patient and to detect method-specific characteristics, 37 AML-, 3 MDS- and 6 healthy MNC were cultured in parallel in 5 different DC-media. This means that only those cases qualified for this analysis where all 5 media (MCM Mimic, Ca Ionophore, Picibanil, Cytokines and Poly (I:C)) had been tested in parallel.

4.3.1 Similar average amounts of mature, viable, migratory and leukemia-derived DC can be generated from AML, MDS and healthy MNC fractions under different parallel culture conditions

Comparing yields of DCsubtypes, we found similar average results as gained by pooling results from all methods (p-value always > 0.05).In AML samples (n=43) especially average amounts of DCopt (on average between 27(±18)% DCopt after culture with MCM Mimic and

30(±19)% after culture with Poly (I:C)) were very similar after culture with 5 different methods (figure 15a). Yields of mature DC ranged from 43(±21)% (Picibanil) to 52(±23)% (MCM Mimic). Highest amounts of DCleu were found after culture with Ca Ionophore

(60±23%), whereas yields of DCleu were lower after culture with MCM Mimic (on average

45±26%) or Cytokines (47±30%). Average amounts of viable DC always were ≥ 63% in AML samples (figure 16a). Yields of Blacon ranged from 31(±23)% (Ca Ionophore) to

38(±22)% (MCM Mimic) in AML samples (figure 15a).

A parallel comparison of average DC amounts after culture was possible in only 3 cases of MDS: yields of DCopt ranged from 19(±12)% (Ca Ionophore) to 27(±10)% (Poly I:C) (figure

15b). Highest numbers of migratory DC were found after culture with Ca Ionophore and Cytokines (50%). Yields of mature DC ranged from 45(±19)% (MCM Mimic) to 65(±28)% (Cytokines). Highest numbers of viable DC and DCleu were found for MCM Mimic (70±29%

and 60±6%). Yields of Blacon in cultured MDS samples ranged from 24(±12)% (MCM

Figure 15: Quantification of DC subsets in AML (a) and MDS (b) after culture, parallel comparison of 5 different methods: DC were generated from blast-containing AML and MDS MNC using 6 different DC-generating methods. Quantification of different subsets of DC was performed after culture using combinations of DC, costimulatory, maturation and migration antigens. Average results (mean± standard deviation) are given. Proportions of flow

cytometrically estimated DCopt, migratory DC (CCR7+), mature DC (CD83+), viable

(7AAD-), DCleu and Blacon were analyzed in AML (n=137) and MDS (n=49) samples. DCopt

optimum of DC, i.e. for each individual sample, the DCA with lowest expression on naïve cells and with highest expression rates after culture was evaluated; DCopt means the

percentage of cells that expressed the specific DCopt marker after culture (percentage of cells

in the total cell amount). (a)

Figure 15 (b)

DCopt optimum of DC. DCleu leukemia-derived DC. Blacon converted blasts.

4.3.2 Similar average expression of specific DCA can be found on DC from AML MNC under different, parallel cultured conditions

To compare the average expression of DCA+ cells for the given 5 methods that were tested in parallel, average expression rates of all available DCA for every single method were pooled. It could be shown that average counts of DC generated with different DC-generating methods were similar in AML samples (10% after culture with Ca Ionophore, 12% after culture with MCM Mimic and Poly I:C, and 13% after culture with Picibanil and Cytokines) (data not shown), whereas DCA were expressed in lower degrees on healthy samples (between 5% after culture with Ca Ionophore and 9% after culture with Cytokines) (data not shown). An analysis of average DCA expression rates on MDS samples was not possible due to a low case number.

Moreover, it is demonstrated in figure 16 that average expression rates of selected DCA in AML cases that were evaluated after parallel culture are similar, although individual

variations of expressions occur. Average expression rates of <10% after culture with any of the 5 methods could be demonstrated for CD1a, CD25 and CD137L. Highest expression rates

were found for CD1b (between 24% after culture with Ca Ionophore and 30% after culture with Cytokines). No data were available for CD83.

Due to only 3 MDS cases and 6 healthy samples available for parallel comparison, a differential analysis of DCA expression rates under different culture conditions was not possible.

Figure 16: Average proportions of single DCA expressed on AML cells after culture; parallel comparison of 5 different methods: only AML (n=37) were included where all 5 methods (MCM Mimic, Ca Ionophore, Picibanil, Cytokines and Poly (I:C)) had been tested in parallel to generate DC. Average expression rates (mean) of 10 selected DCA, depending on methods used, are given. Only cases were included with less than 7% DCA+ cells before culture.

4.3.3 Average gain of DCA+ cells on DC generated from AML, MDS and healthy MNC is similar under different parallel culture conditions

The gain of DCA+ cells after culture in AML MNC after parallel culture in the 5 different media was compared. Again, cases with more than 7% DCA+ cells before culture were excluded. Figure 17 shows comparable gains of DCA+ cells in AML-samples after parallel culture in different DC-differentiating methods with highest gain of DCA+ cells found for CD1b (between 2471% after culture with Ca Ionophore and 4269% after culture with Cytokines) (figure 17). It could be demonstrated that on average, all tested DCA are

upregulated after culture, independent from the methods used for DC generation; however, in some individual cases, a downregulation of one or more DCA could be found after culture. No data were available for CD83.

Due to only 3 MDS cases and 6 healthy samples available for parallel comparison, a

differential analysis of DCA upregulation under different culture conditions was not possible.

Figure 17: Gain of DCA+ cells in AML cases after culture; parallel comparison of 5 different methods: only AML (n=37) were included where all 5 methods (MCM Mimic, Ca Ionophore, Picibanil, Cytokines and Poly (I:C)) had been tested in parallel to generate DC. Average gain (mean) of 10 selected DCA, depending on methods used, are given. Only cases were included with less than 7% DCA+ cells before culture.